1. Field of Use
The present invention is related to electric equipments used in electrophoresis, specifically to the generation of contour clamped electric potentials for generating homogeneous fields that alternate its direction of application.
2. Discussion of the Background and Prior Art
The Electrophoresis
The electrophoresis is a technique that separates molecules by their differential migration inside an electric field. The molecules can be placed in a gel and are sieved when the electric field that compels them to migrate is applied. The negative charged molecules migrate toward the anode and the positive charged ones make it toward the cathode. This way the molecules are separated in bands inside the gel, according to their size. For the generation of the electric field, two parallel electrodes connected to a direct current power supply are usually disposed.
DNA molecules are negatively charged when they are dissolved in buffer at neuter or alkaline pH. When the electric field is applied, DNA molecules are elongated and their charge-mass ratio becomes independent of its molecular size. The above mentioned reasons, together to the fact that the DNA molecules migrate through the pores of the gel in a similar way to the movement of a snake, that is to say by means of a reptation mechanism, it determines that the molecules bigger than 20000 base pairs cannot be separated in electrophoresis at constant electric field, even when they are subjected to molecular sieving.
Pulsed Field Gel Electrophoresis
Pulsed field gel electrophoresis (PFGE) was created by Schwartz and Cantor in 1984 (Cell, 37, pp 67-75, 1984; U.S. Pat. No. 4,473,452 of Sep. 25, 1984) and it increased the range of the DNA molecules that could be separated in electrophoresis. The authors obtained that the large intact DNA molecules, larger than 20000 base pairs, were separated in band patterns inside agarose gels by means of the application of electric pulses of selected duration that periodically alternated their direction of application regarding the separation gel. The changes in the direction of the electric field application cause reorientation of the DNA molecules migration, while the duration of this reorientation depends on the molecular size. The resulting band patterns have been denominated ‘electrophoretic patterns’, ‘molecular kariotypes’, ‘electrophoretic kariotypes’, etc.
This way, any system of pulsed field gel electrophoresis consists of:
1. The electrophoresis chamber with their accessories
2. The appropriate electronics to alternate the electric fields with the desired intensity and pulse duration.
3. The method for polarizing the electrodes.
The electric fields that were generated in the initial PFGE equipments, such as those described by Schwartz and Cantor (Cell, 37, pp 67-75, 1984; U.S. Pat. No. 4,473,452 of Sep. 25, 1984) and others as those described by Carle and Olson (Carle G. F., Olson M. V. Nucleic. Acid Res., 12, pp 5647-5664, 1984) they didn't offer homogeneous values of intensity of the electric field along the gel, so the trajectory and the migration velocity of the DNA molecules in this gels depended on the position that they occupied inside the gel.
Generation of Homogeneous Electric Fields in PFGE.
In theory, two infinite electrodes placed in parallel and separated to certain distance generate a homogeneous electric field. But the design of such electrophoresis chamber is impracticable. To approach to the obtaining of an electric field of homogeneous intensity along the separation gel using finite electrodes, Chu (Chu G., Vollrath D., Davis R. W. Science, 234, pp 1582-1985, 1986) proposed the following:
1. A regular polygon is selected (square, rectangle or hexagon) as a closed contour upon whose sides an array of electrodes will be placed to generate inside the polygon an electric field of homogeneous intensity values.
2. The ‘X’ axis (y=0) of an imaginary Cartesian plane is made coincide with one of the sides of the regular polygon.
3. A 0 volts potential is applied to those electrodes placed at y=0
4. A ‘V0’ volts potential is applied to the electrodes placed at the opposed side of the regular polygon that are at a distance y=A from the ‘X’ axis.
5. In the remaining electrodes, located on the other sides of the regular polygon and at a distance ‘yi’ from the ‘X’ axis, a potential ‘V(yi)’ is applied, where V(yi)=V0·yi/A.
6. This way, the potential generated inside the regular polygon is similar to the one that would be generated by two infinite and parallel electrodes separated a distance ‘A’ one to each other.
7. If the polarity of the electrodes placed at two pairs of opposed sides is electronically exchanged an angle among the lines of force of the resulting electric fields will be form. This angle is denominated in PFGE ‘reorientation angle’.
8. The reorientation angle obtained when the polarity among the electrodes of two different pairs of sides is electronically exchanged will be 90° in the square and 60° or 120° in the hexagon.
The hexagonal configuration of the electrodes array has been the one mostly used in the current systems of PFGE. Said system was denominated Contour Clamped Homogeneous Electric Field or CHEF and it was introduced by Chu in 1986 (Chu G. Science 234, pp 1582-1585, Dec. 16, 1986).
One of the deficiencies of the current CHEF system is that the closed contour of electrodes is limited to the regular polygons previously described.
Methods to Clamp the Voltages in the Electrodes of the CHEF System and to Obtain Electric Fields of Homogeneous Intensity Inside the Gel
Three methods have been mainly proposed, they were gaining in complexity and electronic components:
1. A simple voltage divider (Chu G., Vollrath D., Davis R. W. Science, 234, pp 1582-1585, 1986).
2. The voltage divider associated to transistor pairs in push-pull configuration (Maule J., Green D. K. Anal. Biochem. 191, pp 390-395, 1990).
3. The use of operational amplifiers to control better the voltages imposed in each electrode of the CHEF system (Clark S. M., Lai E., Birren B. W., Hood L. Science 241, pp 1203-1205, 1988).
The Simple Voltage Divider in the PFGE Systems
One of the methods to clamp the potential values in the CHEF electrodes is to use a network of resistors that are connected in series. This network forms a voltage divider among the values zero and ‘V0’. We will name nodes to the place of union between two serial resistors of the voltage divider and at each node is connected an electrode of the hexagon.
The electrodes placed in y=0 and y=A, that is to say two opposed sides of the hexagon are connected to the potentials ‘0’ and ‘V0’, respectively. There are two other groups of electrodes; the electrodes of two consecutive sides of the hexagon form each group. Each one of those electrodes is connected to a node of the voltage divider that defines the potential that should be applied in this electrode. The potential value that is imposed is calculated like it was mentioned in the previous paragraph. For that reason, the two electrodes that are in two different sides of the hexagon, but they are at the same distance ‘yi’ from the more electronegative electrodes (y=0), they should be at the same voltage value given by V(yi)=V0·yi/A.
To achieve the change in the application direction of the electric field, which is indispensable in PFGE, the potential difference is applied to other two different groups of electrodes. This is carried out with relays and diodes which connect the electrodes that should be polarized with zero volt and ‘V0’ to the outputs of the power supply through the system for the electric fields switching.
However, the use of series of resistors to clamp the voltages has an inconvenience. When the network of resistors and the buffer solution came into contact, the latter behaves as a new resistor connected in parallel with the resistors of the network. The currents that are injected from the resistors toward the electrodes and vice versa change the value of the potential in each electrode and affect the electric field homogeneity. The voltage change depends on the amount of current that is injected to or it is extracted from the buffer solution which in turn depends on changes in the concentration, temperature, volume and pH of the buffer solution, among others. These changes affect randomly the conductivity of the buffer and therefore the magnitude of the electric current that is exchanged with the pure resistive circuit (Maule J., Green D. K. Anal. Biochem. 191, pp 390-395, 1990). These random changes in the voltage patterns are uncontrollable and therefore, they affect in a different way the results and the reproducibility of the electrophoretic patterns that are obtained in each experiment.
Those changes can be reduced if the current passing trough the series of resistors is much bigger than the one which circulates by the buffer (Maule, J. and Green, D. K. Anal. Biochem. 191, pp 390-395, 1990). However, that solution has the disadvantage that it causes an unnecessary waste of electric power and forces to use components (especially the resistors) of higher power that are more expensive.
The Voltage Divider Associated to Pairs of Transistors in Push-Pull Configuration
To solve the problems outlined for the resistive voltage divider the use of current sources made of semiconductor elements was proposed (Maule J., Green D. K. Anal. Biochem. 191, pp 390-395, 1990). Those current sources separate each electrode from their corresponding node in the series of resistors of the divider. Between each node and their corresponding electrode a pair of transistors is placed in the configuration called ‘push-pull’. They inject to and extract electric current from each electrode, then repeating in the electrodes the voltage from the node of the divider without has been affected by the changes of conductivity of the buffer solution. The mentioned system is able to polarize the electrodes appropriately in the two directions of application of the electric field in PFGE. However, it has some limitations:
1. The pairs of electrode that should be polarized with same voltage value, V(yi)=V0·yi/A, gets its potential from different nodes, therefore, the equality of voltages in all required electrode pairs is not always achieved.
2. The electrodes nearer the more electropositive electrodes receive the electric current from the NPN type transistor of the push-pull they are connected to. While the electrodes nearer the more electronegative electrodes sink electric current toward the PNP type transistor of the push-pull they are connected to. The fact that transistors of different polarities are active at the same time introduces errors in the pattern of voltages.3. The resistors that set the potential pattern in one of the two direction of application of the field are the same ones that make it in the other direction. For that reason, it is not possible to make independent adjustment of the potentials pattern in each field. Any variation wanted to be introduced in one of the two directions necessarily affects the other direction.4. The circuit has as many transistor pairs in push-pull configuration as electrodes has the CHEF chamber. The transistor pairs in push-pull configuration are connected in parallel. When some of the transistors get broken it is difficult to determine the damaged pair.5. In the transistors pairs configured in push-pull one of the transistors it is always active while the other one is inactive. This means that in all moment half of the transistors are inactive. However, those transistors cannot be eliminated from the circuit, because when the electric field is applied in the other direction, some pairs change the active transistor. Therefore, the voltage divider network connected to transistors pairs in push-pull configuration is inefficient, since the total number of transistors inactive in each field is excessive the same as the total quantity of transistors.6. All the transistor pairs are connected to the power supply without any element that limits the current. The failure of a single transistor causes short circuit between the positive and negative outputs of the power supply. So, it can be concluded that the circuit is not safe.The Use of Operational Amplifiers to Control Better the Voltages Imposed in Each Electrode of the CHEF System
Other more complex systems use operational amplifiers to carry out an individual control of the potential imposed in each electrode of the hexagonal array of the chamber (U.S. Pat. No. 5,084,157). Those systems are able to vary the angle between the two directions of application of the electric field but by means of increasing the electronic complexity of the systems, as much in their construction as their operation. Additionally, the elements that carry out the control of the potentials cannot be properly isolated from the power elements. It is necessary the digital conversion what implies new complexities and the cost of the equipment increases.
On the other hand, Riverón and cols. (Cuban patent, application No. 2000-306) demonstrated that for obtaining straight a reproducible band patterns in PFGE is necessary to guarantee electric fields of homogeneous intensity inside the electrophoresis chamber. They determined that the homogeneity of the applied electric field can be only obtained if, besides having a system for the proper polarization of the electrodes in the closed contour, the electric resistance homogeneity of the buffer and the gel is guaranteed. If the electric resistance is described asR=(1/σ)·(d/A)where: (σ) it is the conductivity of the electrolyte, (d) it is the separation among the electrodes of opposed polarities and (A) it is the cross section area to the flow of the electric current.
It is deduced that for the electric resistance was homogeneous in the whole chamber it is necessary that turbulent flow does not exist in the buffer surface neither deformations nor meniscuses in the gel that alter or modify the cross section area to the flow of the electric current.
Therefore, if PFGE systems, still those that have very complex electronic circuits to polarize the electrodes, do not assure the homogeneity of the buffer electric resistance, they cannot guarantee straight band patterns and reproducible experiments. This situation becomes more critics with small chambers.